This application relates to a method, reagent and kit for evaluating susceptibility to and causation of premature atherosclerosis and other forms of coronary artery disease. The invention further relates to a method of gene therapy by which lipoprotein lipase deficiencies can be treated, and to transducing vectors for use in such a method.
xe2x80x9cCoronary artery diseasexe2x80x9d is a collective term for a variety of symptomatic conditions including angina, myocardial infarction, and nonspecific chest, arm and face pain, which result from atherosclerosis of the arteries that supply blood to the heart. Atherosclerosis, commonly known as xe2x80x9chardening of the arteriesxe2x80x9d is caused by the formation of deposits of fatty substances such as cholesterol within the inner layers or endothelium of the arteries.
xe2x80x9cPremature atherosclerosisxe2x80x9d as used herein refers to the clinical presentation of signs and symptoms of coronary artery disease before the age of 65.
Because of the significant relationship between coronary artery disease and heart attacks, considerable effort has been devoted to identifying the biochemical causes of atherosclerosis. This research has shown that high levels of total cholesterol, low density lipoprotein (LDL), very low density lipoprotein (VLDL) and triglycerides are associated with increased risk of coronary artery disease, while high levels of high density lipoproteins (HDL) are associated with decreased risk of coronary artery disease. See. Gordon et al., The Amer. J. Med. 62: 707-714 (1977). However, while observation of lipoproteins, cholesterol and triglycerides can provide a basis for identifying individuals at risk of coronary artery disease, the levels of these substances are themselves symptoms of an underlying biochemical defect which remains unidentified. Thus, specific treatment of the ultimate cause rather than an intermediate condition, and prediction of risk prior to the onset of this intermediate condition is not possible through such observation.
Studies directed towards the underlying cause of coronary artery disease have identified a number of mutations in genes coding for proteins involved in lipid transport and metabolism that appear to be associated with an increased risk. Examples include a large number of mutations in the low-density lipoprotein receptor gene, Hobbs et al., Human Mutations 1: 445-466 (1992), and a single mutation in the apolipoprotein-B (Apo-B) gene which underlies familial defective Apo-B in many parts of the world. Soria et al., Proc. Nat""l Acad. Sci. USA 86: 587-91 (1989). In addition, mutations in other genes which play a significant role in HDL metabolism such as the cholesterol ester transferase protein (CETP) gene, Brown et al., Nature 342: 448-451 (1989) and the gene for Apo-A1, Rubin et al., Nature 353: 265-266 (1991), have also been shown to be associated with either enhanced resistance or increased susceptibility to atherosclerosis. However, these mutations are uncommon and thus far no specific mutation in any gene has been found in a significant number (i.e.,  greater than 1%) of patients with coronary artery disease or premature atherosclerosis. Accordingly, these test results while interesting do not offer the opportunity to provide evaluation or therapy to significant numbers of patients
It has now been found that a single point mutation in the human lipoprotein lipase gene which results in an Axe2x86x92G nucleotide change at codon 291 (nucleotide 1127) of the lipoprotein lipase gene, and a substitution of serine for the normal asparagine in the lipoprotein lipase gene product is seen with increased frequency in patients with coronary artery disease, and is associated with an increased susceptibility to coronary artery disease, including in particular premature atherosclerosis. This is expressed as a diminished catalytic activity of lipoprotein lipase, lower HDL-cholesterol levels and higher triglyceride levels. Thus, in accordance with the present invention there is provided a method for evaluating susceptibility of a human individual to premature atherosclerosis and other forms of coronary artery disease comprising the steps of:
(a) obtaining a sample of DNA from the individual; and
(b) evaluating the sample of DNA for the presence of nucleotides encoding a serine residue as amino acid 291 of the lipoprotein lipase gene product. The presence of a serine residue is indicative of increased susceptibility in the patient.
The invention further provides a kit for performing the method of the invention. Such a kit comprises a pair of primers selected to amplify a region of a human lipoprotein lipase gene spanning amino acid 291 of human lipoprotein lipase. Appropriate additional reagents may also be included in the kit such as polymerase enzymes, nucleoside stock solutions and the like.
A further aspect of the present invention is a method of treating patients suffering from or likely to suffer from premature atherosclerosis and other forms of coronary artery disease as a result of a lipoprotein lipase deficiency using gene therapy. This may be accomplished using adenovirus-mediated or retrovirus-mediated gene therapy, and can be performed using either an in vivo or an ex vivo approach.